Journal of General Microbiology (1987), 133, 2447-2456.
Printed in Great Britain
2447
Different Molecular Forms of Invertase in the slime Variant of Neurospora
crassu: Comparison with the Wild-type Strain
B y M A N U E L C A S A N O V A , JOSE P . M A R T I N E Z , M. L U I S A G I L ,
R A F A E L S E N T A N D R E U * A N D JOSE R U I Z - H E R R E R A
Departament de Microbiologia, Facultat de Farmacia, Universitat de Valencia, A vgda. Blasco
Ibaiiez 13, 46010- Valencia, Spain
(Received 27 October 1986; revised 6 March 1987)
Invertase synthesis, regulation and secretion in the wall-less slime variant of Neurospora crassa
was studied. Unlike the wild-type, synthesis of the enzyme was not repressed by glucose. This
effect was not related to the 0s mutation harboured by the slime strain, nor to the phenotypic
absence of a cell wall. Three molecular forms of extracellular invertase, which varied in size,
were detected in the slime strain. These forms were interconvertible, with the equilibrium in
favour of the larger form. Polypeptide analysis of the three separated forms revealed that all
contained the same glycoprotein with an M , of 97000. This was completely deglycosylated by
treatment with endo-P-N-acetylglucosaminidaseH (Endo H) to a polypeptide with an M , of
72000. It was concluded that the three interconvertible forms correspond to the monomeric,
dimeric and tetrameric states of the enzyme. Three similar forms of invertase, albeit of slightly
different electrophoretic mobility, were found in cell-free extracts, cell walls and spent culture
medium of the wild-type strain. After Endo H treatment, analysis showed that these forms
contained a polypeptide that was equally reactive against anti-Saccharomyces cerevisiae
antibodies, and had the same M,, as the polypeptide produced by the slime strain.
INTRODUCTION
The slime strain of Neurospora crassa lacks a defined cell wall because of its incapacity to
synthesize the structural polysaccharides chitin and glucan (Leal-Morales & Ruiz-Herrera,
1985). This strain contains normal or higher amounts of chitin synthase (Selitrennikoff, 1979;
Bartnicki-Garcia et al., 1984; Leal-Morales & Ruiz-Herrera, 1985) compared to the wild-type
strain, but has no detectable glucan synthase activity (Leal-Morales & Ruiz-Herrera, 1985). It
has been hypothesized that the normal mechanisms for delivery to the cell surface or for
activation of both transglycosidases are inefficient in this strain (Leal-Morales & Ruiz-Herrera,
1985). On the other hand, secretion of invertase (Bigger et al., 1972) and several other
extracellular glycoproteins (J. P. Martinez, M. Casanova, M. L. Gil, H. Rico, R. Sentandreu &
J. Ruiz-Herrera, unpublished) seems to be unaffected in this strain. These observations suggest
that synthesis and transport of structural polysaccharides and extracellular glycoproteins occur
independently from each other.
To gain information on how these routes of macromolecule externalization operate, the study
of invertase secretion in the slime strain appears attractive. A considerable body of information
on invertase regulation, structure and gene control exists for Saccharomyces cerevisiae (Gascon et
al., 1968; Perlman & Halvorson, 1981 ; Carlson & Botstein, 1982), but data for the N . crassa
system are very scant and relate to the walled wild-type strain only, in which the enzyme is
localized, as in S. cerevisiae, in the periplasmic space (Metzenberg, 1963a, b; 1964; Sargent &
Abbreuiarion : P M S F , phenylmethylsulphonyl fluoride.
0001-3793 0 1987 SGM
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M . CASANOVA A N D OTHERS
Woodward, 1969; Meachum et al., 1971 ; Bigger & Braymer, 1975). However, nothing is known
about the invertase produced by the wall-less slime strain.
In this work we studied the regulation of invertase secretion in the slime strain and compared
the results to invertase secretion in wild-type N . crussu.
METHODS
Strains, culture media and growth conditions. N . crassa wild-type strain FGSC 988 (St Lawrence 74-OR8-la), the
0s-1 B-135 mutant FGSC 951 and the wall-less slime strain FGSC 11 18 were used throughout this work. Vogel's N
medium (Vogel, 1964)supplemented with different sugars (2%, w/v, final concentration), nutrient broth (0.75%),
and yeast extract (0.75%) (pH 5.8) was normally used to grow the wild-type strain and the 0s-I mutant. For growth
of the slime strain and 0s-I protoplasts, this medium was supplemented with 1 M-sorbitol.This is a modification of
Scarborough's 'diet' medium (Scarborough, 1975), and is described in the text as S D M medium. The wild-type
strain and 0s-1 mutant were maintained on 1.5% (w/v) agar slants of Vogel's N medium supplemented with 2%
(w/v) sucrose and 0.06 pg biotin ml-l. The slime strain was maintained on 1.5% agar slants of S D M medium, and
subcultured every 1&15 d.
Strains were propagated in liquid media essentially as described by Leal-Mordles & Ruiz-Herrera (1985). For
short incubation periods in order to measure invertase secretion, cells were harvested by centrifugation, washed
once with 1.2 M-sorbitol, and transferred to the appropriate fresh medium.
The 0s-1 mutant was propagated as a protoplast population following a procedure modified from that of
Selitrennikoff et al. (1981). An Erlenmeyer flask (250 ml) containing 10 ml filter-sterilized Vogel's N medium
supplemented with 1.5% sucrose, 10% (w/v) sorbose, 0.02 mg biotin ml-I, 0.4 mg arginine ml-' and 0-4mg
nikkomycin ml-l was inoculated with lo5 microconidia ml-l, and incubated at 37 "C with gentle shaking. After
48 h incubation the culture was filtered through glasswool to remove mycelium. Fresh medium (10 ml) was then
added to the filtered culture which was incubated again at 37 "C for 48 h. This step was repeated several times until
a homogeneous population of 0s-1 protoplasts was obtained.
Preparation of cell samples and cell-free extracts. Wild-type and 0s-1 mutant germlings or young mycelia were
harvested by centrifugation at 1000g for 10 min, or by filtration through Whatman no. 41 paper, and washed with
the appropriate buffer. Mycelia were transferred to Duran bottles, mixed with glass beads, and shaken in a MSK
cell homogenizer (Braun) for 60 s while the vessel was cooled with a stream of liquid C 0 2 .Cell walls from the wildtype strain were sedimented from cell-free extracts at 1000g for 10 min, thoroughly washed by centrifugation with
water, and freeze-dried. slime and 0s-I protoplasts were harvested by centrifugation at l O O O g for 10 min, washed
twice with a solution of 1.2 M-sorbitol, and lysed by resuspension in the appropriate enzyme assay buffer without
osmotic stabilizer.
Determination of enzyme activities. Invertase was determined by the procedure of Gascon & Lampen (1968).
Before assaying invertase activity in samples from the different spent culture medium, the samples were dialysed
for 48 h at 4 "C against 4 x 2 1 10 mM-sodium acetate buffer, pH 4-9. One unit of invertase activity is the amount of
enzyme that released 1.0 pmol glucose min-I. Invertase activity in polyacrylamide gels was detected according to
Gabriel & Wang (1969).
Separation of oligomeric forms of invertase. Chilled absolute ethanol was added to spent culture medium
containing invertase activity to give a final ethanol concentration of 75% (v/v). The mixture was allowed to stand
at 4 "C for 48-72 h. The material precipitated by ethanol was recovered by centrifugation (27000 g, 30 min), dried
under a stream of nitrogen, and resuspended in 50 mM-ammonium acetate buffer, pH 4.9. The resuspension was
clarified by centrifugation at 3000g for 10-15 min. The resulting supernatant was passed through a Sephadex
(3-50 column (1 -6 x 13 cm, gel bed volume 25 ml) equilibrated with 50 mM-ammonium acetate buffer, pH 4.9. The
void volume that eluted from the column and contained the invertase activity was recovered and freeze-dried. The
lyophilized sample was resuspended in ammonium acetate buffer, applied to a Sephadex G-100 column
(1.6 x 70 cm, 140 ml gel bed volume), and eluted with the same buffer. Fractions (1.5 ml) were collected, and
those containing invertase activity were pooled (see below) and freeze-dried. Dried material was resuspended in
small volumes of buffer solution, and subjected to non-denaturing preparative gel electrophoresis (see below). To
recover the different invertase forms separated after electrophoresis, 1 cm longitudinal strips were cut out from
both sides of the gel; one strip was stained for invertase activity whereas the other one was stained with Coomassie
blue. The remainder of the gel was frozen and transverse sections corresponding to the invertase activity as
revealed by the stained strips were cut out, crushed, and extracted for 24 h at 4 "C in 50 mxl-ammonium acetate
buffer. The crushed gel was sedimented by centrifugation, re-extracted as above, centrifuged again, and the
supernatants obtained after both extractions were pooled and freeze-dried. The different oligomeric forms of
invertase recovered by this procedure were submitted to further analysis by gel electrophoresis under denaturing
or non-denaturing conditions, before or after endo-P-N-acetylglucosaminidaseH (Endo H) treatment.
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Ini>ertase in the slime strain of N . crassa
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Cell-wall-bound inrertase from the wild-type strain. Cell-wall-bound invertase was extracted from purified cell
walls by digestion of the wall glucan network with Zymolyase 20T [50 pg Zymolyase 20T (mg dry wt cell wall)-1],
in a final volume of 1 ml distilled water containing 4 mM-phenylmethylsulphonyl fluoride (PMSF), for 12 h at
28 "C with gentle agitation. The digested cell wall residue was removed by centrifugation, and the supernatant
obtained was freeze-dried. The lyophilized material was resuspended in 50 mM-ammonium acetate buffer, pH 4.9,
and subjected to electrophoretic analysis either under non-denaturing or denaturing conditions.
Treatment with Endo H . Lyophilized invertase samples were treated (at 28 "C with gentle agitation for 48 h) with
Endo H (0.01 units per 10 units of invertase activity, corresponding to about 25-50 pg invertase protein) in 0.1 ml
reaction volumes containing 150 mM-sodium citrate, pH 5.5, 1 mM-PMSF, 10 pM-pepstatin A and 5 mM-sodium
azide. The reaction products were analysed either by denaturing or non-denaturing gel electrophoresis as
described below. In all cases, the samples applied to gels contained about 2 units of invertase activity (5-10 pg
invertase protein).
Pol.vacrylamide gel electrophoresis ( P A G E ) under non-denaturing conditions. Endo-H-treated or untreated
invertase samples (2 vols) were diluted with electrophoresis sample buffer (1 vol. : 35%, v/v, glycerol, 7%, w/v,
SDS and 0.0035% bromophenol blue as tracking dye in 0.5 M-Tris/HCl, pH 6.8), and loaded onto 7-5-15% (w/v)
linear acrylamide gradient slabs (acrylamide :bisacrylamide ratio 30 :0.2) made up in 0.37 ~-Tris/HClbuffer,
pH 8.8, plus 0.1 % SDS. The running buffer system used was 25 m~-Tris/192mwglycine, pH 8.3, containing 0.1%
SDS. Electrophoresis was done at a constant current (30 mA per slab) at 4 "C until the tracking dye front was
0.5 cm from the bottom edge of the gel. For analytical purposes 1.5 mm thick gels were used, whereas preparative
gels were 3 mm thick.
Gel electrophoresis under denaturing conditions (SDS-PAGE) and blotting. Proteins were separated using the
discontinuous buffer system of Laemmli ( I 970) in 7-5-1 5 % linear acrylamide gradient gels (acrylamide :bisacrylamide ratio 30:0-2). Samples were treated with the same sample buffer system as described above but
containing 14% (v/v) 2-mercaptoethanol (sample : solubilizing solution, 2 : 1, v/v) and heated for 5 min in a boiling
water bath before being loaded onto the gels. For estimation of M,,under denaturing or non-denaturing
conditions, gels were calibrated by running in parallel the following M , markers : myosin (205000), b-galactosidase
(1 16000), phosphorylase b (97000), bovine albumin (66000), ovalbumin (45000) and carbonic anhydrase (29000).
In all cases, the marker proteins were denatured as above.
Electrophoretic transfer to Trans-Blot nitrocellulose paper was as described by Burnette (198 l), except that the
transfer buffer used was 25 mM-Tris/192 mM-glycine, pH 8.3, plus 20% (v/v) methanol, and electrophoretic
transfer was done at 6-8 V cm-* for 15-17 h at 4 "C in a BioRad Trans-Blot cell using an LKB 2197 power supply.
Concanavalin A staining of nitrocellulose blots was done according to Hawkes (1982) with the modifications of
Millette & Scott (1984). Immunochemical detection of proteins on blots was done by following the protocol
described in the BioRad Immun-Blot (GAR-HRP) assay kit based on the procedures of Towbin et al. (1979) and
Burnette (1981), using the specific antibody at a final 1 : 500 dilution in 10 mM-Tris/HCl, pH 7.4, plus 1% bovine
serum albumin, 0.9% NaCl and 0.05% Tween 20.
Miscellaneous. Protein was estimated by the Lowry procedure. Anti-S. cerevisiae invertase antibodies, obtained
as described by Esmon et al. (1981), were kindly provided by S. Mormeneo and J . Zueco of this Department. Gel
electrophoresis and blotting reagents were from Bio-Rad. SDS-PAGE and M , markers were from Sigma. Gel
filtration media (Sephadex G-50 and G-100) were supplied by Pharmacia. Endo H and Zymolyase 20T were from
Miles Laboratories. Nikkomycin was kindly provided by Bayer. Culture media compounds were purchased from
Difco. All other chemicals and reagents were obtained from Sigma or Merck.
RESULTS
Inuertase secretion by the slime strain of N . crassa
Determination of invertase in the medium after growth of the slime strain revealed the
presence of high levels of the enzyme. Cells grown for 48 h in SDM hypertonic medium with
glucose were transferred to fresh medium containing either no sugar, glycerol or raffinose (a
trisaccharide formed by elongation of sucrose with a galactose residue through a glycosidic
bond). Secretion of invertase took place immediately with similar kinetics independently of the
carbon source. After 6 h the extracellular enzyme levels were about 6-fold higher than those
associated with the intact cells, which remained constant throughout this time (Fig. 1).
Cycloheximide (100 pg ml-' ) completely blocked enzyme secretion, thus demonstrating that
secreted enzyme is synthesized de novo. In the presence of tunicamycin (15 pg ml-I) the levels of
active enzyme in the medium were reduced by 95 %. This observation suggests that glycosylation
is necessary for secretion of active invertase.
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M . CASANOVA A N D OTHERS
3
Time (h)
1
5
Fig. 1. Secretion of invertase by the slime strain of N. crassa. Cells grown for 48 h in glucose-containing
SDM medium were transferred to fresh SDM medium containing no sugar (0,.), 2%glucose (V,V),
2% glycerol (0,M) or 2% raffinose a,A).
Open symbols correspond to invertase activity detected in
cell extracts, whereas closed symbols correspond to enzyme activity in the growth medium.
Table 1. Catabolite repression of invertase biosynthesis in the wild-type and 0s-1 mutant of
N . crassa
Spores (lo6 ml-l) were inoculated into SDM (see Methods). Germlings were harvested by
centrifugation, and washed and resuspended in 50 mhl-sodium acetate buffer, pH 4.9. Cell growth is
expressed as mg protein per 50 ml medium. Invertase was measured in whole cells and is expressed as
units (mg protein)-'.
Sugar
added to
medium
None
Glucose (2%)
Wild-type
0s-I with
4% (w/v) NaCl
0s- 1
& & &
Cell
growth
Invertase
Cell
growth
Invertase
Cell
growth
Invertase
5-72
8.78
0.843
0.047
22.6
145-0
2.225
0.070
2.05
16.40
0-324
0.007
Regulation of invertase secretion in several strains of N . crassa
Synthesis of the large form of invertase (extracellular) is subject to catabolite repression in S .
cerevisiae (Carlson & Botstein, 1982). In the presence of glucose almost no enzyme is secreted.
The results reported above showed that glucose had no inhibitory effect on the secretion of the
enzyme in the slime strain of N . crassa (Fig. l), indicating no catabolite repression.
We determined whether lack of catabolite repression was specific for the slime strain or a
general phenomenon in other N . crassa strains. Wild-type conidia were germinated in liquid
media containing different sugars, and extracellular invertase was measured. The results (Table
1) showed that invertase biosynthesis was repressed by glucose.
The N . crassa 0s-I mutant is a strain harbouring one of the mutations present in the slime
strain (Emerson, 1963). Invertase secretion by this strain was also repressed by glucose either in
the absence or in the presence of the concentration of NaCl that allowed limited cell growth and
phenotypic expression of osmotic sensitivity (Table 1).
To determine whether lack of catabolite repression in the slime strain was somehow related to
the absence of the cell wall, the 0s-1 mutant was grown as a protoplast population as described in
Methods. When these protoplasts were transferred to SDM medium with or without glucose,
secretion of invertase was shown to be subject to catabolite repression (Table 2).
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Invertase in the slime strain of N . crassa
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Table 2. EJect ofglucose on invertase secretion by growing protoplasts of N . crassa 0s-l
Protoplasts of the 0s-I mutant, obtained as described in Methods, were harvested, and washed with
1-2M-sorbitof.Samples of the protoplast suspension (about 10 mg cell protein) were transferred to SDM
medium containing 10% (w/v) sorbose instead of sorbitol, and with or without glucose. At the indicated
times, 10 ml samples were withdrawn. Cells were separated from the medium by centrifugation,
washed twice with 1.2 M-sorbitol, and resuspended in 50 mM sodium acetate buffer, pH 4.9, plus 2%
(v/v) Triton X-100. Invertase was measured in the cell extracts and in the dialysed spent medium (see
Methods). Original invertase activity (time zero) was 0.006 units ml-I in the cells and 0.002 units ml-I in
the medium.
Sugar
added
None
Glucose (2%)
Incubation
time (h)
3
7
3
7
Invertase (units ml-I)
c p A - \
Cells
Culture medium
0.032
0-102
0.006
0-007
0.253
1.173
0.004
0-005
Secretion of'd8erent molecular forms of invertase by the slime strain
Gel filtration in a Sephadex G-100 column of the spent culture media revealed that invertase
eluted as a broad ill-defined peak, with maximum activity in the void volume (not shown), thus
suggesting that the enzyme was microheterogeneous.
In a further experiment the spent medium was subjected to gel filtration in a Sephadex G-50
column, and the void volume obtained passed through a column of Sephadex G-100; the
material eluted from the latter column was combined into four pools (Fig. 2, A, B, C and D).
Analysis of these pools by non-denaturing PAGE resulted in the separation of invertase into
three forms with estimated M , values of 76000, 132000 and 235000 (Fig. 2b). The larger form
dissociated into the other forms on subsequent electrophoresis under non-denaturing conditions
(not shown).
DiJerent molecular forms of invertase in the wild-type strain
About 28% of the cell-bound invertase remained firmly attached to the cell-wall fraction of
the wild-type strain. Only a minor part of this invertase could be extracted with neutral
detergents or with urea. However, the enzyme was completely released by treatment with
Zymolyase. Extraction of invertase after digestion of walls with chitinase has also been reported
(Meachum & Braymer, 1969; Sargent & Woodward, 1969; Chung & Trevithick, 1970). Analysis
of the released invertase by non-denaturing PAGE showed mainly two molecular forms with
about the same apparent M , (as revealed by their relative electrophoretic mobility) as forms I
and I1 of the enzyme secreted by the slime strain (Fig. 3, lane A). Similar analysis of the invertase
which, in low amounts, is normally present in the growth medium of young germlings of the
fungus, also revealed the presence of two forms of the enzyme; however, these always exhibited
lower electrophoretic mobilities than the corresponding forms extracted from the cell wall (Fig.
3, lane B).
Molecular structure of N . crassa invertase
The three molecular forms of invertase secreted by the slime strain are glycoprotein in nature,
since treatment with Endo H decreased their M , values proportionally (Fig. 4). In order to
determine the M , values, the nature of the polysaccharide fraction, and the polypeptide
composition of the diffirent forms of invertase, they were separated as described in Methods.
Staining of the non-denaturing preparative gels with Coomassie blue showed the presence of a
single band corresponding to invertase form I, although invertase form 111, and especially form
11, were contaminated with other polypeptides (not shown). SDS-PAGE of the three isolated
invertase forms revealed the presence of the same polypeptide before ( M , 97000) and after ( M ,
72000) Endo H treatment (Fig. 5, lanes A-F) (the lower M , polypeptides present in the tracks
corresponding to invertase forms I1 and I11 come from contaminating proteins).
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M . C A S A N O V A A N D OTHERS
Fig. 2. Chromatographic behaviour of invertase forms secreted into the medium by slime cells. (a)
Ethanol-precipitated culture medium was subjected to chromatography on a Sephadex G-100 column.
(b) Fractions eluted from the column were pooled, freeze-dried, subjected to gel electrophoresis under
non-denaturing conditions, and stained for invertase activity: lane A, fractions eluted from 50-54 ml ;
lane B, 55-58 ml; lane C, 59-63 ml; lane D, 64-72 ml. The different oligomeric forms (I, 11, 111) of
invertase detected in the gel are indicated.
Treatment of the different invertase forms from the slime strain with Endo H left a
polypeptide which did not react with Concanavalin A after blotting to nitrocellulose sheets (Fig.
6 a , lane B). This observation indicates that treatment with Endo H completely removed the
carbohydrate moiety of the molecule. Both glycosylated and deglycosylatedpolypeptides reacted
with antibodies raised against S. cerevisiae invertase (Fig. 6 a , lanes C and D). The M , of the
glycosylated polypeptide was calculated to be 97000 (Fig. 5, lane A); that of the deglycosylated
polypeptide was 72000 (Fig. 5 , lane B).
Anti-S. cerevisiae invertase antibodies were used to detect the invertase forms from wild-type
N . crassa in nitrocellulose blots after SDS-PAGE. Invertase forms obtained from the culture
medium and the periplasmic space (not shown), and that released from the cell walls by
digestion with Zymolyase, all gave the same polypeptide after Endo H digestion, with an M ,
identical to that of the polypeptide from the invertase forms secreted by the slime strain (Fig. 6b,
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Invertase in the slime strain of N . crassa
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Fig. 3
Fig. 4
Fig. 3. Invertase forms present in the cell walls (lane A) and the growth medium (lane B) of wild-type N.
crassa. Separation was achieved by non-denaturing gel electrophoresis. Bands were detected by
invertase activity staining. Arrows marked I, 11 and I11 indicate the mobilities of the corresponding
invertase forms secreted by the slime strain.
Fig. 4. Change in electrophoretic mobility (non-denaturing conditions) after Endo H treatment of
invertase forms secreted by the slime strain. Invertase forms were from pool A (see Fig. 2) after (lane A)
or before (lane C) Endo H treatment, or from pool D (see Fig. 2) after treatment with Endo H (lane B).
The gel was stained for invertase activity.
Fig. 5. Polypeptide composition of the three invertase forms secreted by the slime strain. Invertase
forms I, 11, and 111 were separated and treated with Endo H as described in Methods. Electrophoretic
analysis was done under denaturing conditions and polypeptides were detected by Coomassie blue
staining. Lanes A, C and E, untreated invertase forms I, I1 and 111; lanes B, D and F, electrophoretic
pattern of invertase forms I, I1 and 111 after Endo H treatment.
lanes B and D). The glycosylated invertase polypeptide from the wild-type strain appeared to be
more heterogeneous and to have a higher M , (Fig. 6b, lane C) than that from the slime strain
(Fig. 6b, lane A).
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M . C A S A N O V A A N D OTHERS
Fig. 6. (a) Samples of slime invertase form I untreated, or treated with Endo H as indicated, were
subjected to SDS-PAGE, transferred to nitrocellulose blotting media and reacted with Concanavalin A
(lanes A and B) or antibodies raised against S . cerevisiae invertase (lanes C and D). (b) Reactivity
towards anti-S. cerevisiae invertase antibodies (immunoblotting) of slime invertase form I (lanes A and
B), and invertase (untreated, or treated with Endo H as indicated) released by Zymolyase from the cell
wall of the wild-type strain (lanes C and D).
DISCUSSION
We confirmed that invertase is secreted by the slime strain of N . crassa as originally reported
by Bigger et al. (1972), but we found that secretion occurred continuously without the lag phase
described by these authors. Complete inhibition of invertase biosynthesis and secretion by
cycloheximide demonstrated that the secreted enzyme is synthesized de nouo, whereas inhibition
of invertase biosynthesis by tunicamycin is evidence of the importance of the carbohydrate
moiety of the enzyme for its translocation, secretion, and probably its stability.
Synthesis and secretion of invertase by the slime strain occurred independently of the carbon
source present in the culture medium, whereas catabolite repression was normal in the wild-type
strain. Synthesis of invertase in S. cerevisiae is subject to catabolite repression (Carlson &
Botstein, 1982), but there exist catabolite-resistant mutants which exhibit an increased rate of
invertase synthesis and secretion (Carlson et al., 1984). Resistance to catabolite repression in the
slime strain was not correlated with the 0s mutation, one of the three mutations necessary to
maintain the slime phenotype (Emerson, 1963). Thus, an 0s-2 mutant showed normal repression
when grown in either normal or hypertonic media. Neither was the lack of control of
biosynthesis and secretion of the enzyme the result of the phenotypic loss of the wall, since
invertase secretion by the 0s-2 mutant growing as a protoplast population was repressed by
glucose as normal. Equally important was the finding that the same molecular forms of invertase
were detected in the spent medium of cells grown either in the presence or in the absence of
glucose. Catabolite repression mutants of S. cerevisiae hex1 and hex2 are affected in hexokinase
PI1 (Entian & Frohlich, 1984). Whether a similar mutation occurs in slime strains remains to be
determined. Anyhow, this trait must be added to the three mutations known to be harboured by
the slime cells (Emerson, 1963). However, it should be mentioned that the absence of catabolite
repression of invertase production in the slime strain is not the result of a general phenomenon of
alteration in the regulatory mechanisms, since synthesis of exocellular phosphatases is still
repressed by phosphate (unpublished observations).
Metzenberg (1964) described the presence of two interconvertible forms of invertase in the
periplasmic space of wild-type N . crassa: a heavy one (s20,w= 10-3) and a light one
(s20,w= 5.2). He concluded that the larger form was a tetramer of the light form. These data
were confirmed by Meachum et al. (1971). In this study we found three interconvertible forms of
invertase in the slime strain. Apparent M , values of the three forms under non-denaturing
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Inuertase in the slime strain of N . crassa
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conditions were 76000, 132000 and 235000. Similarly, we found three forms of invertase, albeit
with slightly different M , values, in the periplasmic space, bound to the cell wall, and in the
culture medium, of the N . crussa wild-type strain. Cell-wall-bound invertase was tightly
associated with the glucan microfibrillar network, and was mostly released only after glucan
digestion with Zymolyase.
Multimeric forms with a predominance of the octamer have been reported for S. cerevisiae
invertase (Chu et al., 1983). Interestingly, the monomeric form has no catalytic activity (Chu et
ul., 1983). This phenomenon, plus the close relationship between invertase from S.cerevisiue and
N . crussa revealed by the cross-reaction of the latter with antibodies raised against the yeast
enzyme, prompted us to investigate whether or not the low M , form with invertase activity from
the N . crussa slime strain represented the monomeric form of the enzyme. Determination of the
M , of the polypeptide from the three forms of invertase secreted by the slime cells by SDS-PAGE
gave a value of 97000; the M , of the deglycosylated polypeptide was 72000. These values are
close enough to the M , of the light active form I11 observed under non-denaturing conditions.
Regardless of the discrepancies in mobility that are due to the different electrophoretic
conditions used in each case (see below), this result suggests that the monomeric form of the
enzyme is active. The intermediate (11) form appears to be the dimer and the heaviest one (I) the
tetramer. Their respective M , values would then be 194000 and 388000 respectively. The
disparity between these values and those obtained by non-denaturing PAGE is due to the
abnormal mobilities of polypeptides under these conditions. The M , calculated for the monomer
differs from the value (51 500) described by Meachum et al. (1971) for wild-type N. crassu. The
dissimilarity may be due to the fact that these authors used centrifugation in the presence of
guanidine chloride to determine M,, a method which may be largely dependent on molecular
shape.
Treatment of all the invertase forms from the slime strain with Endo H gave the same
polypeptide ( M , 72000). This polypeptide failed to react with Concanavalin A, an indication
that all the carbohydrate was bound to the polypeptide moiety of invertase through N-glycosidic
bonds. The difference in M , between the glycosylated and deglycosylated polypeptide was
25000. Accordingly, the sugar moiety in each invertase subunit represents about 25 % of the M,,
a value about twice that reported by Meachum et al. (1971) for invertase from the wild-type
strain (14%). The same M , was calculated for the deglycosylated polypeptide from wild-type N .
crassa. This indicates that the invertase from the two N . crassa strains contains the same
polypeptide, and that differences in the M , values of the glycosylated polypeptides from
different invertase fractions are probably due to the different sizes of the carbohydrate moiety of
the molecules. Cross-reaction of N. crassa invertase with antibodies directed against the enzyme
from S. cerevisiue (whose polypeptide moiety is significantly smaller: estimated M , 60000;
Trimble & Maley, 1977) implies conservation of a significant portion of the molecule, and its
relationship with the suc2 gene product of the yeast suc family (Carlson & Botstein, 1983).
This work was partially supported by grants no. 3432-83 of the Comision Asesora de Investigacion Cientifica y
Tecnologica (Spain) and 400/85 from COSNET (Subsecretaria de Educacion e Investigacion Tecnologicas,
Mexico). J. R.-H.
is an Investigador Nacional from Mexico, and was on leave from Instituto de Investigacion en
Biologia Experimental, Universidad de Guanajuato, and Centro de Investigacion y Estudios Avanzados from the
Instituto Politecnico Nacional, Mexico; his stay in Spain was defrayed through CAICyT (Spain) and CONACYT
and COSNET (Mexico).
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